1. Field of the Invention
The present invention relates to a microscope suitable for high-throughput screening comprising an autofocusing apparatus having an unfolded, main optical axis. Further the invention also relates to an autofocusing apparatus and to an autofocusing method useful in high-throughput screening.
2. Description of the Related Art
Autofocusing techniques for microscopes have been available for many years.
Most autofocus methods fall into two categories: position sensing and image content analysis. Image content autofocus functions have previously been compared for brightfield microscopy. Groen, Young and Ligthart (Groen FCA, Young IT, Ligthart G: A comparison of different focus functions for use in autofocus algorithms. Cytometry 6:81-91, 1985) compared 11 autofocus functions under brightfield using an electron microscope grid and a metaphase spread, and Vollath (Vollath D: Automatic Focusing by Correlative Methods. J Microsc 147:279-288, 1987) tested an autocorrelation function under brightfield using a paralytic steel specimen. Groen et al. concluded that three autofocus functions, i.e., two gradient functions and the intensity variance, performed the best. Its most important limitation is speed, which is dependent on the calculation performances.
In a typical autofocusing image content technique, an objective lens is placed at a predetermined distance from the sample to be scanned, and an image is taken of the object. The image created by the microscope is then typically evaluated to determine the position at which the surface of the object, or a plane within the object, is in focus. The evaluation of the image typically involves analyzing characteristics of the image such as entropy, spatial resolution, spatial frequency, contrast, or other characteristics. The analysis of these characteristics requires a considerable amount of computer processing. Once the characteristics are analyzed, the distance between the objective lens and the object to be scanned is varied, and another image is taken. The new image is then evaluated and the process is repeated several times before a focused image is finally obtained. Repeating the step of analyzing the image may cause the focusing operation to take an undesirably long time before the microscope is finally focused on the object surface. The need for increased processing time for autofocusing can be particularly acute for various types of imaging operations. For example, when an object is observed under a microscope, the focused conditions must be maintained in order to maintain a properly focused image of the object. Therefore, even if the object is initially in focus, the object may gradually become out of focus due to a variety of external factors such as thermal effects and vibration, if no corrective steps are taken. Moreover, when an object is larger than the field of view of the microscope, the microscope can only focus on the portion of the object that can be observed through the field of view of the microscope. Therefore, the focusing conditions must be regularly checked and adjusted In order to maintain a sharp image of the whole object.
In view of the foregoing, there is a need for an improved autofocusing system and method for a microscope that can perform quick and accurate autofocusing operations while maintaining a sharp image.
The present invention is in a first aspect related to a microscope having autofocus position sensing means useful in high-throughput screening. The uncertainty in applying autofocus test results from one microscope method to another led to the present invention. The development of the present invention included exploring autofocus performance in microscopy of fluorescent stained biologic specimens.
Several autofocus position sensing methods and apparatuses are known, for example from Offenlegungsschrift DE 34 46 727 and DE 33 28 821. These German documents disclose an autofocus device for a microscope wherein variations in light intensity originating from two separate light sources provide a signal for focus adjustment. These known autofocus methods are in particular useful for flat samples to be imaged. The autofocusing light beams travel along a substantial large part including several optical elements, such as a plurality of lenses and at least two beam splitters. This large part causes a substantial delay in the autofocusing procedure. The present invention is directed to a more simplified autofocus system whereby the part of autofocusing light is minimized. The uncertainty in applying autofocus test results from one microscope method to another led to the present invention. The development of the present invention included exploring autofocus performance in microscopy of fluorescent stained biologic specimens.